This application relates to enzymatic synthesis of compounds, particularly low molecular weight peptides containing from 2 to 10 amino acid residues. It relates specifically to the synthesis of alpha-L-aspartyl-L-phenylalanine lower alkyl esters.
One of such esters, the methyl ester (hereinafter APM), is commonly known as aspartame, a powerful sweetening agent. It is comprised of L-phenylalanyl methyl ester linked through a peptide bond to an L-aspartyl residue.
APM has been synthesized by many methods, including directly reacting L-phenylalanine methyl ester and an N-protected aspartic acid anhydride, or by enzymatically joining N-protected L-aspartic acid and an L-phenylalanine methyl ester. A modification of this enzymatic method, which does not require the use of N-protected L-aspartic acid, is further described by Harada et al., EPA 74,095. In the method of Harada et al. a culture of certain enumerated microorganisms is contacted with L-aspartic acid and a methyl ester of L-phenylalanine in order to synthesize APM. In the Examples, the microorganisms were cultured and suspended in a medium containing L-aspartic acid and L-phenylalanine methyl ester, APM was allowed to accumulate, the cells were separated from the medium and the supernatant medium thereafter was subjected to fractionation. The APM recovery ranged from 1.2 to 7.5 g (adjusted on the basis of one liter of culture medium). Molar yields ranged from 0.5 percent to 1.4 percent based on the amount of phenylalanine methyl ester added to the reaction mixture.
The peptide yields obtainable by the Harada et al. enzymatic synthesis are limited by the equilibrium of the enzyme-catalyzed reaction, which tends to favor the amino acid reactants rather than the dipeptide product. Extensive efforts have been devoted to improving the equilibrium in favor of the synthetic product (Oyama et al., "Chemtech", February 1984, pp 100-105). Sparingly soluble peptidyl products are favored in the equilibrium, but not all peptides have such characteristics or, if such characteristics are introduced, they may be difficult and expensive to remove from the final product. For example, carbobenzoxylated (N-blocked) L-aspartic acid and L-phenylalanine methyl ester have been enzymatically conjugated to yield an insoluble addition compound (Isowa et al., U.S. Pat. No. 4,436,925). This method is disclosed to result in product yields of up to 99.1 percent at the immediate conclusion of fermentative synthesis. However, additional steps are required to remove the benzoylcarbonyl moiety and the potential exists for product contamination by the L-aspartic acid derivative.
Water-miscible or immiscible organic solvents have been used in attempts to improve the yields of synthetic protease reactions. These methods are unsatisfactory because many organic solvents inhibit protease activity and the products must be separated from the solvent by expensive processes. Also, the solvent can be costly, and must be efficiently recycled.
Notwithstanding such efforts to secure highly efficient synthesis of contaminant-free products, the art has failed to assemble an economical system for enzymatic peptide synthesis. Heretofore, the art has necessarily traded-off increased yields in the enzyme catalyzed step against requirements for further processing of the reaction product, including the removal of potentially toxic substances. Accordingly, the objects of this invention include synthesizing peptides in high yield from ordinary microbial cultures or immobilized enzymes, but without the need either to later remove product substituents or otherwise undertake covalent modifications of the product peptide, or to purify the product from organic solvents. These and other objects of this invention will be apparent from consideration of this application as a whole.